Reporter proteins are usually used as markers for visualizing in vivo gene expression and protein translocation in eucaryotic and prokaryotic organisms. The most widely used reporter proteins in plants may be exemplified by β-glucuronidase (GUS) and luciferase (LUC). Particularly, GUS is a prevalent visualization marker in the plant cell biology (Jefferson R A, et al., (1987), EMBO J., 6:3901-3907). However, histochemical GUC analysis is not suitable for the direct visual selection of transgenic plants per se because it requires the destruction of plant tissues. As for LUC, its in vivo synthesis can be detected; however, an external substrate, that is luciferin, is required for the detection of LUC. In addition, LUC has the disadvantage of emitting a low intensity of light (Ow D W et al., Science 234:856-859).
Recently, it has been reported that the green fluorescent protein (GFP) of jellyfish Aquorea Victoria can be utilized as a sensitive reporter for in vivo gene expression (Chalfie M., et al., 1994, Science 263:802-805). Even though requiring no external factors, the detection of the fluorescence generated from an isolated GFP is possible only with near infrared (386 nm) or blue light (475 nm). However, the fluorescence of the GFP can be observed under visible light in room conditions (Chalfie M., et al., 1994, Science 263:802-805; Delagrave S., et al., 1995, Bio/Technology 13:151-154; Heim R, et al., 1994, Proc Nat'l, Acad Sci. USA, 94:2122-2127). Further, GFP model is very advantageous in that it retains fluorescence even when being expressed in heterologous biosystems, such as E. coli, yeast, Drosophila, insects, mammals, etc. (Brand A 1995, TIG 11:324-325; Chalfie M., et al., 1994, Science 263:802-805; Cubitt A B, et al., 1995, TIBS 20:448-455; Davis S J, et al., 1998, Plant Mol. Biol. 36:521-528; Delagrave S, et al., 1995, Bio/Technology 13:151-154; Haseloff J., et al., 1997, Proc. Nat'l Acad. Sci. USA, 94:2122-2127; Rosario R., et al., 1995, Curr. Biol. 5:635-642; Wang S X et al., 1994, Nature 369:400-403).
Bioassays for gene expression using fluorescence are very useful for monitoring the transformation and growth of plants. Because of plant tissues' being composed of highly reflective cell walls and aqueous cytoplasm containing various autofluorescent and light-scattering materials, it is difficult to directly observe the exogenous fluorescence of proteins introduced into plant tissues under a fluorescent optical microscope (Haseloff J. et al., 1998, Green fluorescent protein: Properties, applications, and protocols. Chalfie M., Kain S., Eds., Wiley-Liss, New York, pp 191-242). For this reason, the use of GFP as a marker for the selection of transgenic plants under direct visual conditions has not yet been reported.